Retroviruses are enveloped, single-stranded RNA viruses whose replication depends on the integration of a double-stranded DNA intermediate, termed the provirus, into the host cell genome. Onco-retroviruses, such as the murine leukemia virus (MuLV), depend on cell proliferation for completion of their life cycle (Humphries and Temin, Journal of Virology 10:82-87 (1972); and Humphries and Temin, Journal of Virology 14:531-546 (1974)). The breakdown of the nuclear envelope that accompanies mitosis is essential to bring the MuLV preintegration complex into the vicinity of the host cell chromosome, allowing integration of the viral genome into the host cell chromosome (Lewis and Emerman, Journal of Virology 68:510-516 (1994); and Roe et al., EMBO Journal 12:2099-2108 (1993)).
In sharp contrast, lentiviruses (including HIV) are distinguished by their ability to infect non-dividing cells. For instance, HIV-1 replicates in terminally differentiated tissue macrophages (Meltzer et al., Annual Review of Immunology 8:169-194 (1990)), as well as in cells that are artificially arrested in the G1/S or G2 phases of the cell cycle (Lewis and Emerman supra; and Lewis et al., EMBO Journal 11:3053-3058 (1992)). A critical determinant of this property has been mapped to the HIV-1 matrix (MA) protein, one of the virus structural components encoded by the gag gene (Bukrinsky et al., Nature 365:666-669 (1993)).
MA is the N-myristoylated cleavage product of the HIV-1 p55 Gag precursor by the virally-encoded protease. MA plays a key role in retroviral assembly, by directing the intracellular transport and membrane association of the Gag polyprotein (Varmus and Swanstrom, In: RNA Tumor Viruses, Weiss, et al., eds (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) 369-512 (1982); Yuan et al., Journal of Virology 67:6387-6394 (1993); Zhou et al., Journal of Virology 68:2556-2569 (1994); and Facke et al., Journal of Virology 67:4972-4980 (1993)) , and by facilitating the recruitment of the viral envelope into viral particles (Yu et al., Journal of Virology 66:5966-5971 (1992); and Dorfman et al., Journal of Virology 68:1689-1696 (1994)). MA also appears to participate in the early steps of the viral life cycle (Yu et al., Journal of Virology 66:5667-5670 (1992)).
In lentiviruses, MA contains a stretch of highly conserved amino acids, for example, Gly-Lys-Lys-Lys-Tyr-Lys (SEQ ID NO:l) in HIV-1 (Myers et al. eds. Human Retroviruses and AIDS 1992: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences (Los Alamos National Laboratory, Los Alamos, N.M.) (1992)). This conserved sequence closely resembles the SV40 large T antigen nuclear localization signal (NLS) (Kalderon et al., Cell 39:499-509 (1984)). Indeed, the HIV-1 MA NLS peptide has been shown to act as a nuclear import signal when coupled to a heterologous protein in vitro (Bukrinsky supra). In addition, an HIV-1 strain mutated in the MA-NLS-coding sequence could not infect cells arrested in the cell cycle, thus displaying a phenotype more characteristic of an onco-retrovirus (Bukrinsky supra). The defect was associated with a failure to induce viral two-LTR circular DNA in growth-arrested cells. Because this form of DNA is generated only after the viral preintegration complex migrates to the nucleus, this result indicated further that the MA NLS plays a role in the infection process. These findings corroborated the earlier detection of MA in purified HIV-1 preintegration complexes (Bukrinsky et al., Proc. Natl. Acad. Sci. U.S.A. 90:6125-6129 (1993)) as well as in the nucleus of newly infected cells (Sharova and Bukrinskaya., AIDS Research and Human Retroviruses 7:303-306 (1991)), and led to the postulate that the HIV-1 MA NLS might be critical for infection of macrophages. Indeed, this has recently been demonstrated by the showing that HIV-1 MA NLS mutants replicate with normal kinetics in dividing cells, including activated peripheral blood lymphocytes (PBL), but do not grow efficiently in terminally differentiated primary macrophages. Furthermore, it has recently been discovered that MA NLS mutants cannot establish a stable and inducible infection intermediate in unstimulated PBL (von Schwedler et al., Proc. Natl. Acad. Sci. U.S.A. 91:6992-6996 (1994)).
For infection of non-dividing cells, the HIV-1 preintegration complex must cross the nuclear envelope. Most but not all proteins migrating to the nucleus contain amino acid sequences at least vaguely related to the archetypal nuclear localization signal (NLS) of SV40 large T antigen NLS (i.e., PKKKRKV; SEQ ID NO:2; see Dingwall and Laskey, TIPS 16:478-481 (1991)). Transport of macromolecules across nuclear envelope can be divided into two distinct steps (reviewed in Forbes, D. J., Annual Review of Cell Biology 8:495-527 (1992)). The initial reaction includes the formation of a complex between the karyophile and a cytoplasmic NLS receptor, or Nu-transferon (Varshavsky, A., Cell 64:13-15 (1991)). Putative NLS receptors have been identified by NLS ligand binding and crosslinking studies, as well as through the functional reconstitution of import NLS-containing karyophiles in permeabilized cell assays (Adam and Gerace, Cell 66:837-847 (1991); Liet al., Experimental Cell Research 202:355-365 (1992); Okuno et al., Experimental Cell Research 206:134-142 (1993); Pandey and Parnaik, Biochimica et Biophysica ACTA 1063:81-89 (1991); and Stochaj and Silver, Journal of Cell Biology 117:473-482 (1992)). None of these receptors, however, has been cloned.
NLS-mediated nuclear import can be kinetically saturated by microinjecting high concentrations of synthetic T-antigen NLS peptide. NLS peptide competes the nuclear migration of a large number of cellular karyophiles, indicating that most of these are imported by a single pathway (Michaud and Goldfarb, Experimental Cell Research 208:128-136 (1993)). Nuclear migration of the HIV-1 preintegration complex obeys the same rules, in being an energy dependent process which can be competed with NLS peptide (Bukrinsky et al., Proc. Natl. Acad. Sci. U.S.A. 89:6580-6584 (1992); and Gulizia et al., Journal of Virology 68:2021-2025 (1994)); this corroborates the demonstrated role of the MAN LS in this process.
The mechanisms developed by HIV-1 to infect non-dividing cells are most likely essential for the spread of this virus in infected individuals, and for AIDS pathogenesis. As such, the various steps involved in the completion of this process represent highly suitable targets for the development of novel antiviral therapies. In addition, the resulting knowledge might have broader implications, in the field of gene therapy. Indeed, the stable expression of foreign genes is often desirable in cells which enter mitosis at a low frequency (such as myocytes or even hepatocytes), or do not divide at all (such as macrophages and neurons).
Because conventional retroviral vectors cannot efficiently infect such targets, alternatives are being sought. Non-retroviral vectors are one option. Adenovirus-based vectors are promising, but their failure to stably integrate into the genome of target cells limits their use, because transgenes are only transiently expressed. In contrast, vectors derived from adeno-associated virus do integrate; however, it has so far been extremely difficult to generate high titer stocks of these vectors, whereas this is a prerequisite for efficient gene transfer. Alternative strategies have therefore been developed to allow the use of simple retroviral vectors in cells which rarely divide.
For instance, partial hepatectomies are performed to enhance the uptake of MuLV-based vectors by regenerating hepatocytes. Also, cells such as myocytes are induced to proliferate ex vivo, infected with retroviral vectors while dividing, and then re-implanted. However, such techniques cannot be applied in a number of cell types, such as neurons. Furthermore, even when genes are efficiently transferred, a major problem remains that most often their expression is only transient. Although the mechanism of this silencing is yet unclear, one can speculate that the reorganization of the chromatin, which takes place when a cell passes from a dividing to a resting state, plays a critical role in this process. Indeed, it could be that regions of the genome in which retroviruses preferentially integrate, when cells are dividing, subsequently become transcriptional "cold-spots" when they stop proliferating.
Accordingly, it might be preferable to transfer genes in a context reproducing the in vivo state of the cell. This could be done if retroviral vectors with the properties of lentiviruses were available. In view of these considerations, the lessons learned from studying HIV-1 infection of non-dividing targets are expected to one day help make very significant progress in the field of gene therapy. Ironically, one area which might directly benefit from this progress is that of approaches based on anti-HIV intracellular immunization, where one requirement is to reach the same non-dividing cells which can be infected by the virus itself.